AN ELECTRON MICROSCOPE STUDY OF CALF THYMUS NUCLEAR PREPARATIONS ISOLATED IN SUCROSE SOLUTIONS

Abstract

This work was carried out with the intent of developing a method capable of routinely evaluating calf thymus nuclear preparations with the electron microscope. Examination of small random samples, pre-embedded in agar after fixation with permanganate, were found to give results comparable to those obtained with much larger samples withdrawn randomly from pellets and embedded and sectioned conventionally. Results obtained by this pre-embedding technique with acrolein, osmium tetroxide, or permanganate fixations were equivalent. Calf thymus nuclear preparations isolated in sucrose by the method prevalently used (see 1) are contaminated only slightly with intact cells, to a degree which varies with each preparation. However, intact cells, damaged cells, or nuclei with some cytoplasm constitute together about 30 per cent of the preparation. Particles other than intact cells are not readily distinguishable from one another by light or phase microscope techniques. These preparations can be purified further by centrifuging through a dense sucrose layer. In our hands, however, contamination with some cytoplasm still remains in approximately 10 per cent of the particles. Incubation of the particles prepared without purification procedures, under conditions frequently used, results in the extensive breakdown of particles. Under at least one set of conditions, nuclei are selectively disrupted, leaving primarily damaged cells in the preparation.